Antineoplastic Preparation and the Use of Antineoplastic Preparation

ABSTRACT

A method for manufacturing an antineoplastic preparation containing alpha-ketoglutarate (AKG) or/and glutamine or/and glutamate or/and alpha-ketoglutarate of ornithine or/and dipeptides of glutamine and other amino acids or/and tripeptides of glutamine and other amino acids or/and di- and tripeptides of glutamate with other amino acid, or/and mono- and divalent metal salts and other of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-ketoglutarate.

An object of the invention is antineoplastic preparation and the use ofantineoplastic preparation.

In spite of the significant progress in the chemotherapy, radiotherapyand immunotherapy in last years the problem of the effectiveantineoplastic therapy is still the serious challenge in a presentmedicine. Epidemiological research indicates that in countries fullydeveloped one of three people suffers from the different neoplasticdiseases. In that group each fourth case is a lethal one. Cytostaticsactually used in treatment are at the risk of side effects occurencelimiting the effectiveness and decreasing the quality of patients life.

Therefore the development of new drugs acting specifically on theneoplastic cells with the simultaneous protective activity on the normalcells is especially urgent challenge.

The antineoplastic preparation distinguishes itself that containsalpha-ketoglutarate (AKG) or/and glutamine or/and glutamate or/andalpha-ketoglutarate of ornithine or/and dipeptides of glutamine andother amino acids or/and tripeptides of glutamine and other amino acidsor/and di- and tripeptides of glutamate with other amino acid or/andmono- and divalent metal salts and other of alpha-keto-glutarate or/andglutamine or/and glutamate or/and ornithine of alpha-ketoglutarate.

The use of antineoplastic preparation is that the preparation containingalpha-ketoglutarate (AKG) or/and glutamine or/and glutamate or/andalpha-ketoglutarate of ornithine or and dipeptides of glutamine andother amino acids or/and tripeptides of glutamine and other amino acidsor/and di- and tripeptides of glutamate with other amino acid or/andmono- and divalent metal salts and other of alpha-keto-glutarate or/andglutamine or/and glutamate or/and ornithine of alpha-ketoglutarate isused in the prophylaxis of neoplastic diseases.

Other use of antineoplastic preparation is that the preparationcontaining alpha-ketoglutarate (AKG) or/and glutamine or/and glutamateor/and alpha-ketoglutarate of ornithine or/and dipeptides of glutamineand other amino acids or/and tripeptides of glutamine and other aminoacids or/and di- and tripeptides of glutamate with other amino acidor/and mono- and divalent metal salts and other of alpha-keto-glutarateor/and glutamine or/and glutamate or/and ornithine ofalpha-keto-glutarate is used for metastases inhibition.

The preparation and the use of the preparation lets for the inhibitionof the migration of neoplastic cells reflecting the potential role ofAKG in the metastases inhibition. The preparation added to the dietplays a role as neo-adjuvant supporting existing methods used in theneoplasm treatment. It may improve the quality of the patients lifethrough the synergic action with antineoplastic drugs and simultaneousprotective activity to normal cells.

The example of the preparation action and its use is shown as a way ofinvention executing:

FIG. 1—the curve presenting the proliferation of A549 cells with thestimulation by AKG,

FIG. 2—the proliferation of C6 cells with AKG and

FIG. 3—the proliferation of HT-29 cells with AKG stimulation, whereas

FIG. 4 presents the proliferation of human neoplastic cells A549 in thepresence of the cyclophosphamide with AKG, while

FIG. 5—the proliferation of human neoplastic cells A549 in the presenceof iphosphamide with AKG,

FIG. 6—the proliferation of human neoplastic cells A549 in the presenceof thiotepa with AKG,

FIG. 7—the inhibition of C6 cells migration due to AKG.

The cultures of neoplastic cells:

A549—human neoplastic cells of the lung cancer; the continuous lineobtained from the Institute of the Immunology and the ExperimentalTherapy of Polish Academy of Science in Wroclaw

HT-29—human neoplastic cells of the large intestine cancer; thecontinuous line obtained from the Institute of the Immunology and theExperimental Therapy of Polish Academy of Science in Wroclaw

C6—the rat neoplastic cells of the brain cancer (glioma); the continuousline obtained from the Department of Neonatology, Humboldt University,Berlin, Germany

The bases of the culture.

Cells of A549 line were cultured on the basis DMEM:F-12 HAM (2:1), HT-29and C6 cells on the basis DMEM. To the culture basis 10% foetal beastserum (FBS), penicillin 100 i.u./ml and streptomycin 100 μg/ml wereadded. The bases DMEM:F-12 HAM, DMEM were produced by Sigma company(Sigma, St. Louis, Mo., U.S.A.). The foetal beast serum (FBS) wasproduced by Life Technologies company (Life Technologies, Karlsruhe,Germany). Remaining reagents were produced by Sigma company.

The preparation of cellular cultures.

Cells stored in a liquid nitrogen in a tissue bank were unfreezed in atemp. 37° C., then poured into the plastic bottles containing properbasis. They were cultured in a temperature 37° C. in the incubator with5% CO2 flow. After cells reproduction liquid was poured out, cells werewashed with PBS (without the calcium and magnesium ions) and processedwith 0.25% trypsin solution+EDTA to receive the suspension of cellsnecessary in the experience.

Assessment of the antyproliferative activity of AKG in the cellularculture.

Prepared earlier, in the culture basis, suspension of cells with adensity 1×10⁴ cells/ml (A549), 4×10⁴ cells/ml (HT-29) and 0.5×10⁴cells/ml (C6) were poured into the 96-pits microplate with the flatbottom (NUNC company, Roskilde, Denmark) in the volume 100 μl/pit. Aftersticking of cells (24 hours) the liquid was carefully pulled down andthen different concentrations of AKG and examined cytostatics(cyclophosphamide, iphosphamid, thiotepa) in liquid with 10% FBS (100μl/pit) were added. The cultures on plates were sleft for 96 hoursincubation in temp. 37° C., in the atmosphere 95% air and 5% CO2. Theantyproliferative activity of examined substances was assessed with themethod MTT.

The method MTT (according to kit “Cell proliferation kit III”,Boehringer Manheim).

This method was worked out to determine the proliferation and vitalityof cells in studies on the cytotoxic and antyproliferative substances.In metabolically active cells tetrazolic yellow salt MTT is reduced toformazane blue with the mitochondrial dehydrases. Formazane crystals,insoluble in water, accumulate in cells and for their dissolutions theuse of organic detergent, breaking the membrane and simultaneouslysolvent the dye, is necessary. For this purpose the buffer SDS-HCl withpH 7.4 is used. The concentration of released dye is evaluatedquantitatively in the reader for 96-pits plates at the wavelength 570nm. The colour intensity is directly proportional to the quantity ofalive cells.

MTT solution in PBS condition in concentration 5 mg/ml was added intoeach pit on plastic plate in a dose 15 μl/pit. Plates were incubated for3 hours in temp. 37° C. Thereafter buffer SDS-HCl in a dose 100 μl/pitwas added and plates were left in temp. 37° C. all night. The resultswere evaluated next day with the use of E-max Reader (Molecular DevicesCorporation, Menlo Park, Calif., U.S.A.).

Evaluation of the Cells Migration Degree with the Method “Wound Assay”

This method is for estimation the activity of substances affecting themodility of cells in vitro. It is used in research on wounds healing,angiogenesis and neoplastic metastases.

C6 cells (1×10⁶) suspended in the culture basis with the addition of 10%serum (FBS) were poured onto the culture plates (NUNC, Roskilde,Denmark) with 4 cm diameter. Next day in the equal layer of cells theflaw (wound) was done with the ending of automatic pipette and unstickedcells were removed by twice-rinsing of plates with PBS solution. ThenAKG (10 and 20 mM) dissolved in the culture basis was added to theprepared culture. Plates were incubated for 24 hours in a temperature37° C. and in a humid atmosphere 95% air and 5% CO₂. Thereafter thecultures were coloured with May-Grünwald-Giemza method. Then themicroscopic analysis was performed with the microscope Olympus BX51(Olympus Optical CO., LTD, Tokyo, Japan) with the use of the softwareanalySIS® (Soft Imaging System GmbH, Münster, Germany). The degree ofcells migration was assessed in cytometry as the number of cells whichpopulated the wound done earlier in the layer of cells. At least 50chosen fields on 8 photographs were assessed.

Results: The Estimation of Antyproliferative Activity of AKG

Antyproliferative activity of AKG in the cultures was estimated indifferent kind of neoplastic cells: the lung cancer cells (A549), thelarge intestine cancer cells (HT-29) and glioma cells (C6). Cells wereprocessed with AKG in concentrations 0.5, 1, 2.5, 5, 10 and 20 mM, for96 hours.

The examined substance has had antyproliferative activity with relationto all neoplastic cells types (the diagram—FIG. 1, FIG. 2, FIG. 3).Statistically significant (4.5%) inhibition of cells growth was observedat AKG concentration 2.5 mM in A549 cell line in the comparison to thecontrol group. That effect was correlated with a dose of AKG and was7.8%, 12.4%, 17.5% with doses 5 mM, 10 mM and 20 mM respectively. (thediagram on FIG. 1). The growth inhibition in glioma cells (C6) was 12.6%with AKG dose 2.5 mM, 7.9%—5 mM, 16%—10 nmM and 19.8%—20 mMrespectively. The growth inhibition of large intestine cancer cells(HT-29) hadn't had the lineal character with AKG concentrations between1 and 10 mM. The dose 1 mM inhidited the cells growth by 11.8%. Thesimilar effect was obtained with a dose 5 mM (11.8%) and 10 mM (11.5%).Only a dose 20 mM caused the significant (25%) inhibition of these cellsgrowth (the diagram—FIG. 3).

The estimation of the interaction between AKG and antineoplastic drugs.

The research on the interaction between AKG and popular cytostatics usedin cancer chemotherapy was performed in the culture of lung cancer cells(A549). For that purpose cells were treated with the followingcytostatics: cyclophosphamide (1.5 mM), iphosphamid (1.5 mM) andthiotepa (5 μM), alone and in combination with AKG (5, 10 and 20 mM).The additive effect of AKG on cytostatic activity of usedchemotherapeutics was observed (results are presented on FIG. 4, FIG. 5and FIG. 6). Cyclophosphamide in a concentration 1.5 mM inhibited thegrowth of A549 cells by 21.4%. The addition of AKG increased itscytostatic activity by 6.4%, 9.8% and 14.4% respectively (thediagram—FIG. 4). Iphosphamid (1.5 mM) inhidited the cells growth by7.3%. After AKG addition (5, 10 and 20 mM) that effect increased by 5%,5.3% and 8.8% respectively (the diagram—FIG. 5). AKG intensified alsocytostatic activity of thiotepa (5 μM—25.9%) by 4.2%(5 mM), 8% (10 mM)and 11.2% (20 mM) (results are presented on FIG. 6).

The Influence of AKG on the Migration of Neoplastic Cells

The research on the mobility of neoplastic cells was passed in the“wound assay” model. At the photograph 1 the wound in the layer of C6cells (A), the wound population with cells after 24 hours of incubationwithout AKG (B) and significant inhibition of cells migration in thepresence of AKG 20 mM (C) are presented. On the diagram—FIG. 7, theaverage number of cells migrating to one field of flaw done in theuniform layer of cells is presented. Statistically significantinhibition of cells migration in the presence of 10 mM and 20 mM AKG wasshown.

The statistical analysis

The statistical analysis was performed with the use of t-student test. *p<0.05, ** p<0.01, ***p<0.001.

1. A method for the manufacture of a pharmaceutical preparation for thetreatment or prophylaxis of neoplastic diseases, comprising the step ofpreparing an antineoplastic preparation using ketoglutarate (AKG), mono-or divalent metal salts of alpha-ketoglutarate and/or ornithine ofalpha-ketoglutarate as the only active ingredients.
 2. A method for themanufacture of a pharmaceutical preparation for inhibition of cancermetastases, comprising the step of preparing an antineoplasticpreparation using alpha-ketoglutarate (AKG), mono- or divalent metalsalts of alpha-ketoglutarate and/or ornithine of alpha-ketoglutarate asthe only active ingredients.